Blood is a fluid that contains a variety of cells. The most numerous cells (more than 90%) are the erythrocytes, or red blood cells, which carry out the exchange of oxygen and carbon dioxide between the lungs and the body tissues. The minor population of cells are the leucocytes, or white blood cells, which control the immuno response system of the body and defend the body against infecting organisms and foreign agents both in the tissues and the bloodstream.
The leucocyte population in blood is further defined by a number of subclasses which play distinct roles in the immune response. For example, the relative number cells in various subclasses of lymphocytes (about 20% of the leucocyte population) is likely to change in various disease states. Identification of cells of the various subclasses of lymphocytes provides an indication of the relative well being of the patient.
It is known that at least several particular subclasses of functionally distinct lymphocytes can be identified on the basis of antigenic determinants found on the cell surface. For example, the analysis of the T-cell population of the lymphocyte fraction of leukocytes is currently under intensive investigation in a wide variety of disease states. In renal allograft recipients, for example, monitoring of T-cell subsets in peripheral blood provides information which can be used as a basis for clinical decisions.; Cosimi, A.B., et al., 1981, N. Engl. J. Med. 14,308. Since significance is attached to relatively small changes in the sizes of the subpopulations of T-cells, it is necessary to have an accurate, reproducible method for obtaining data regarding the T-cell subpopulations.
It should be understood that there are two principal classes of lymphocytes involved in the immune system of humans and animals. The first of these (the thymus-derived cell or T-cell) have immunological specificity and are directly involved in cell-mediated immune responses (such as graft rejection). Although T-cells do not secrete antibodies, they are sometimes required for the effective secretion of these antibodies by the second class of lymphocytes discussed below. Some types of T-cells play a regulating function in other aspects of the immune system. The mechanism of this process of cell cooperation is not yet completely understood.
The second class of lymphocytes (the bone marrow-derived cells or B cells) are those which secrete antibody. It is thought that B cells differentiate within the bone marrow.
T-cells are divided into at least several subtypes, termed "helper", "suppressor", and "killer" T-cells, which have the function of (respectively) promoting a reaction, suppressing a reaction, or killing (lysing) foreign cells. These subclasses are well understood for murine systems, and have recently been identified for human systems. The ability to identify subclasses of T-cells is important for diagnosis or treatment of various immunoregulatory disorders or conditions.
Conventional immunofluorescence techniques presently includes the physical separation of the lymphocytes from other leukocytes and the erythrocytes as a preliminary step, usually by density gradient centrifugation (Boyum. A., 1968 Scand. J. Clin. Lab Invest., 21 Suppl. 97). This separation step eliminates the possibility that non-specifically stained monocytes or granulocytes might be counted as specifically stained lymphocytes. This initial lymphocyte isolation step is long and arduous; in fact much longer than the relatively simple step of tagging and analyzing the tagged lymphocytes. The necessity of separating the lymphocytes from other leukocytes and erythrocytes is a serious impediment to rapid clinical analyses. Furthermore, even for research applications, where time is less important, the lymphocyte separation steps involves the risk of loss of some lymphocytes which introduces uncertainty and inaccuracy to the subsequent analysis.
It is, accordingly, a primary object of the present invention to provide a method and lysing agent for identifying and enumerating specific subclasses of lymphocytes in a whole blood sample without the necessity for prior separation of lymphocytes from other blood cells.
It is another object to provide methods and a lysing agent enumerating various subclasses of lymphocytes which are more accurate and precise than present techniques, and which substantially prevent faulty analysis due to the presence of erythrocytes or loss of data through loss of lymphocytes from the sample.
It is a further object to provide such methods wherein the speed and relative simplicity of the method makes lymphocyte subclass identification and enumeration of a viable clinical tool.